Bilirubin-IXAlpha, the principal end product of heme and hemoglobin degradation, is excreted in bile in the form of glucuronide conjugates. In humans and rats, the major bile pigment fraction is bilirubin diglucuronide (BDG). The hepatic mechanism responsible for BDG formation has been in doubt, but we recently have shown that formation of both dilirubin monoglucuronide (BMG) and BDG are UDP-glucuronic acid-dependent reactions catalyzed by the microsomal glucuronyltransferase system. We now propose to define this enzyme system in terms of its properties, regulation and kinetics, and to determine whether a single or multiple enzyme proteins are involved in the synthesis of BDG. Rat and human liver preparations will be used, together with recently developed highly reproducible analytical methods for isolation and quantitation of BMG (C-8 and C-12 isomers) and BDG, and a novel radioassay of bilirubin UDP-glucuronyltransferase (BGT) applicable to human boipsy material. Since BGT is a membrane-bound enzyme system, attention will be paid to the interaction between enzyme function and its lipid environment, which appears to be of importance for enzyme regulation. Liposomes containing bilirubin and partitioning of pigment into microsomal membranes, will be used to determine whether bilirubin reaches the catalytic enzyme site via the lipid phase of the membrane or via the cytosol. This appears to be of functional importance, because BMG, the presumed intermediate in BDG formation, has been found to be a poor substrate for the enzyme when added to the bulk aqueous phase. Since conjugation of bilirubin is required for excretion of the pigment by the liver, detailed information about the conjugation mechanism is essential for understanding the pathogenesis of different forms of jaundice, particularly Gilbert's syndrome, Crigler-Najjar disease and neonatal hyperbilirubinema. THe findings also should clarify why in certain forms of jaundice, BMG is the predominant bile pigment, and why in conjugated hyperbilirubinemia, virtually all the pigment is serum is BMG. Finally, if the genetic deficiency of BGT activity in Gunn rats can be corrected by introducing purified BGT incorporated in liposomes into the liver, this would offer a novel approach of potential therapeutic importance in patients with Crigler-Najjar disease.